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Preclinical PET Imaging of Tumor Cell Death following Therapy Using Gallium-68-Labeled C2Am


Flaviu Bulat et al, Cancers, 2023


There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68Ga-labeled C2A domain of Synaptotagmin-I (68Ga-C2Am), which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68Ga-C2Am (20 min, 25 ◦C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.

Results from nanoScan® PET/CT

PET-CT Imaging of Tumor Cell Death In Vivo Using 68Ga-C2Am

The binding of 68Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vivo using Whole-body PET/CT imaging. COLO205 tumor-bearing mice (N = 6) were scanned consecutively in the same imaging session (total scan time 2.5 h), before and 24 hours following treatment with MEDI3039 (a TRAIL-R2 agonist, Medimmune). CT data was acquired for anatomical reference and for attenuation correction using Nucline (V2.01, Mediso Medical Imaging Systems Ltd.). Images were acquired with a semicircular scan, with 180 projections. The X-ray energy was set to 55 kVp with 600 milliseconds exposure and 1:4 image binning. CT images were reconstructed using Butterworth filtering with an isotropic voxel size of 213 µm. A dynamic PET acquisition lasting 120 minutes was initiated 30 seconds prior to intravenous injection of 3.7 ± 1.2 MBq 68Ga-C2Am (1075 ± 382 µg protein/kg body weight; 10 mL/kg; SA = 0.15 ± 0.05 MBq/µg protein). Scans were reconstructed into 23 time frames (4 between 0–1 min; 4 between 1–5 min; 11 between 5–60 min and 4 between 60–120 min). PET images, with a nominal isotropic resolution of 0.6 mm, were reconstructed with a dynamic protocol using Tera-Tomo 3D PET reconstruction technology (Mediso Medical Imaging Systems Ltd.), the binning parameters were set to energy window 400–600 keV, coincidence modes 1–5, full detector model, normal regularization, 2 iterations, 6 subsets, with attenuation and random scatter correction. Images were analyzed using VivoQuant® software (vs. 4.0 patch 1, InviCRO Ltd.). Standardized uptake values were calculated as maximum (SUVmax) and mean (SUV). SUV was defined Cimg/(ID/BW) where Cimg is the activity concentration (MBq/mL), ID is injected activity (MBq), and BW is body weight (g). SUVmax was calculated from the pixel with the maximum signal intensity.

  • The results obtained by in vivo dynamic PET/CT imaging (Figure 1) confirmed renal as the dominant excretion route for 68Ga-C2Am. Bladder signal could be detected within 15 min of administration (Figure 4a,b), and kidney cortical uptake increased up to 2 hours post-injection (to ~25% ID/g; Figure 4c, green line).
  • The tumor signal was detectable within 15 minutes of injection, and there was a small component (<3% ID/g) of hepatobiliary clearance (Figure 4c, purple line).
  • Tumor-to-muscle (T/m) and tumor-to-blood (T/b) ratios were 2.2 ± 0.2 and 1.1 ± 0.1, respectively, at 2 hours post-administration of 68Ga-C2Am. However, analysis of individual COLO205 tumors before and following treatment (Figure 4d) showed significant increases in tumor retention of 68Ga-C2Am at 2 hours post-administration, expressed as (ID/g, %; p < 0.005), tumor-to-blood (T/b; p < 0.05), SUV or SUVmax (p < 0.005).

Figure 1. PET/CT images of 68Ga-C2Am accumulation in subcutaneous COLO205 tumors (white arrows). (a) Before treatment (BTx) and (b) 24 h after treatment with MEDI3039 at 0.4 mg/kg (Atx). Maximum intensity projections from representative mice. Images were acquired for up to 120 min after intravenous injection of 68Ga-C2Am. Signal (ID/g, %) is overlaid on a skeleton mask derived from CT images. (c) Activities (ID/g, %) in the indicated tissues in drug-treated animals (MEDI3039, 0.1–0.4 mg/kg, 24 h). (d) Pairwise analysis of PET signals before and 24 h after treatment at 2 h after probe administration, expressed as ID/g (%), tumor-to-blood (T/b) ratio, SUV, and SUVmax. Two-tailed, pairwise t-test, * p < 0.05, ** p < 0.01. Data in (c,d) are mean ± SEM, N = 6.

The results obtained by in vivo PET/CT imaging demonstrated 68Ga-C2Am as a PET agent for imaging cell death in vivo. The agent showed a predominantly renal clearance and tumor contrast post-treatment, within 2 h of administration. Considering the wide availability of gallium-68 generators globally, 68Ga-C2Am has the potential to be used in the clinic to assess early response to cancer treatment.

Full article on mdpi.com


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